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Brazilian Journal of Microbiology

Print version ISSN 1517-8382On-line version ISSN 1678-4405

Braz. J. Microbiol. vol.38 no.3 São Paulo July/Sept. 2007

http://dx.doi.org/10.1590/S1517-83822007000300025 

ENVIRONMENTAL MICROBIOLOGY

 

Aeromonas spp. and microbial indicators in raw drinking water source

 

Aeromonas sp. e indicadores microbiológicos em fontes de água não tratada

 

 

Marisa Di Bari; Hachich, Elayse M.; Melo, Adalgisa M.J.; Maria I. Z. Sato*

Companhia de Tecnologia de Saneamento Ambiental, São Paulo, SP, Brasil

 

 


ABSTRACT

Aeromonas species are autochtonous in the aquatic environment and some of them have been associated with health effects like wound infections, septicemia and diarrhoeal illness. In this study, the occurrence of Aeromonas spp. and microbial indicators in raw drinking water from wells, springs, fountains and mineral waters was evaluated. A total of 126 water samples was analyzed for Aeromonas spp. by the membrane filtration technique using ADA media and by P/A test. Typical colonies of Aeromonas spp. were submitted to biochemical tests for species differentiation. Toxin production was tested using Y-1 mouse adrenal cells. Coliforms and heterotrophic bacteria were enumerated by membrane filtration and pour plate techniques, respectively. P. aeruginosa, C. perfringens and fecal streptococci were determined by P/A method. Aeromonas spp. were isolated in 36.5% of the samples, whereas total and thermotolerant coliforms were detected in 51.2% and in 23.8% of the samples, respectively. C. perfringens, fecal streptococci and P. aeruginosa were present in 16.5%, 20.4% and 3.8% of the samples, respectively. The concentrations of heterotrophic bacteria were higher than 1,0x103 CFU/mL in 52.5% of the samples. A. hydrophila was the most frequent species, followed by A. allosaccharophila,A. jandaei,A.sobria and HG2. A heat label toxin was detected in 13 from the 58 strains tested. These data show that the drinking water sources analyzed can represent a risk for human health. It is important to consider that wells and springs are used as drinking water supply in poor areas and rural regions, where undernourished people more susceptible to infections by these microorganisms predominate.

Key-words: Aeromonas, raw drinking water, microbial indicators, heat labile toxin.


RESUMO

Bactérias do gênero Aeromonas são naturais no ambiente aquático e algumas espécies podem causar infecções em humanos como feridas, septicemia e diarréia. Este trabalho objetivou avaliar a ocorrência de Aeromonas sp. em 126 amostras de água de poços, nascentes, fontes e água mineral, e associar sua presença com indicadores microbianos de contaminação. Foi utilizada a técnica de membrana filtrante com o meio ADA e o teste P/A. Colônias típicas de Aeromonas sp. foram submetidas a testes bioquímicos para identificação da espécie. A produção de toxina foi avaliada utilizando-se células Y-1 de adrenal de camundongo. Coliformes e bactérias heterotróficas foram analisados através de filtração em membrana e pela técnica de inoculação em profundidade, respectivamente. P. aeruginosa, C. pefringens e os estreptococos fecais foram determinados pelo teste P/A. Aeromonas sp. foi isolada em 36,5% das amostras, enquanto que os coliformes totais e termotolerantes estavam presentes em 51,2% e 23,8% das amostras, respectivamente. C. perfringens, estreptococos fecais e P. aeruginosa foram detectados em 16,5%, 20,4% e 3,8% das amostras respectivamente. Concentrações de bactérias heterotróficas superiores a 1,0x103 UFC/mL ocorreram em 52,5% das amostras. A. hydrophila foi a espécie mais isolada, seguida por A. allosaccharophila, A. jandaei, A. sobria e HG2. Uma toxina termolábil foi detectada em 13 dos 58 isolados analisados. Portanto, as fontes de água de consumo humano analisadas podem representar um risco para a saúde humana. É importante considerar que fontes, poços e nascentes são utilizadas como suprimento de água em áreas pobres e regiões rurais, onde predominam pessoas com problemas de desnutrição, mais suscetíveis a doenças infecciosas.

Palavras-chave: Aeromonas, água de consumo humano bruta, indicadores microbianos, toxina termolábil.


 

 

INTRODUCTION

The importance of Aeromonas spp. as an emergent human pathogen has increased significantly in the last years (18, 21). Aeromonas spp. can cause wound infections and septicaemia (20,26,29) and have been associated with diarrhoeal illness worldwide (13,16,34). There are presently 22 phenospecies and 18 genomospecies in the genus Aeromonas spp. (24) and at least 9 of them have been implicated in human diseases (18,24). A. hydrophila (HG-1), A. caviae (HG-4), A. veronii (HG-8), A. jandaei (HG-9), A. schubertii (HG-12) and A. trota (HG-14) are more frequently associated with clinical specimens (22,24). Some species are pathogens for fish and amphibians.

The normal habitat of Aeromonas spp. is the aquatic ecosystems and their presence have been reported frequently by several authors in groundwater, drinking water at treatment plants and in water distribution systems, natural mineral springs, surface waters (fresh, estuarine and marine) and crude and treated sewage (1,5,6,8,16,22,23,27,35). Exposure to water contaminated with Aeromonas spp., through ingestion or contact, have been associated with human infections that are particularly hazardous in immunocompromised patients (8,20). Recently, the US Environmental Protection Agency proposed Aeromonas hydrophila as one of the contaminants of concern in waterborne diseases (36).

In the present study the occurrence of Aeromonas species in drinking water proceeding from wells, springs, fountains and mineral waters was investigated. Such drinking waters are usually consumed without any treatment and can be considered as a source of gastrointestinal infections. The prevalence of aeromonads in relation to the microbiological bacterial indicators was also studied.

 

MATERIAL AND METHODS

Sampling

A total of 118 water samples collected over a 12 month period from wells (n=97), springs (n=11) and fountains (n=10) examined for compliance with Brazilian Legislation in the Laboratory of Microbiology of CETESB (São Paulo, SP, Brazil) were selected for this study. Eight samples of mineral water were also examined. These water sources have not been submitted to any antimicrobial treatment. Sample collection was performed according to the APHA Standard Methods (2) in sterile disposable bottles, chilled for storage and transportation. Samples were examined within 24 hours.

Bacteriological Methods

Water samples were assayed for Aeromonas spp. (n=126), total and thermotolerant coliforms (n=126), heterotrophic bacteria (n=120), fecal streptococci (n= 103), Pseudomonas aeruginosa (n=103) and Clostridium perfringens (n=103).

Coliforms were enumerated by the membrane filtration technique and heterotrophic bacteria by the pour plate technique, according to APHA (2).

Fecal streptococci, P.aeruginosa and C. perfringens were determined by P/A assay (9,10). For enumeration of Aeromonas spp. the membrane filtration technique using ampicillin-dextrin agar medium (ADA), developed by Havelaar et al. was used (15). The plates were incubated at 30ºC for 18 to 24 hours. Typical yellow colonies (dextrin fermentation) were counted and five of them were transferred to nutrient agar for oxidase test. Oxidase positive strains were inoculated into IAL medium (28) for an initial screening. At least one presumptive Aeromonas spp isolate of each sample was submitted to the following biochemical tests for genus confirmation and species differentiation (3,30): decarboxylation of lysine and ornithine, dihydrolation of arginine, production of indole, VP reaction, fermentation of glucose, arabinose, inositol, lactose, mannose, mannitol, salicin, sorbitol and sucrose, production of gas from glucose, hydrolysis of aesculin, ONPG and gelatin, susceptibility to O/129 (50 and 150 µg), growth in the absence of sodium chloride, motility and nitrate reduction.

Aeromonas spp. was also ivestigated by P/A tests. Positive P/A presumptive broth was streaked on ADA agar medium and identification was carried out as described above.

Haemolysin production

Haemolysin production was evaluated on 5% sheep blood agar (SBA) as follows: The 4-5h culture of each Aeromonas spp. strain obtained in brain heart infusion broth (BHIB) was inoculated in SBA. After incubation at 35ºC for 24 and 48h the plates were examined for the presence of alpha- or beta- haemolysis (35).

Heat labile (LT) enterotoxin assay

Enterotoxin production was tested in Y-1 mouse adrenal cells according to Sack and Sack (32). Tryptic soy broth containing 0.6% of yeast extract was inoculated with a fresh culture of the Aeromonas spp. strain (BHIB, 4h growth). After incubation at 35ºC for 18h in a shaker, cell free preparations were obtained by centrifugation of the cultures at 10,000xg for 30 minutes at 5ºC followed by filtration through membrane filter (0.45 µm). Supernatant preparations were stored at 20ºC if it was not possible to test the material within 24h. Two aliquots (25 µl each) of the supernatant were inoculated in Y-1 cells (96 well microtitration plates) and the cytotoxic effect was observed after incubation at 37ºC during 6 and 18 hours. Positive and negative controls were performed in parallel with each assay. The heat stability of the samples was tested exposing the supernatants of the positive strains to 56ºC for 15 minutes.

 

RESULTS AND DISCUSSION

The percentage of positive samples for the bacteriological indicators and Aeromonas spp. is shown in Fig. 1. A total of 65 samples out of 126 tested were positive for total coliforms. Thermotolerant coliforms and fecal streptococci were present in more than 20% of the water samples and heterotrophic bacteria were detected in densities higher than 1,0 x 103 CFU/100 mL in 52.5% of the samples. C. perfringens and P. aeruginosa were isolated in 17% and 4% of the samples, respectively. Even lower percentages of positive samples for C. perfringens and P. aeruginosa were obtained by Clark (11).

 

 

Aeromonas species were recovered from 46 samples (36.5%) and the percentage of positive samples was higher in less protected raw drinking water sources, such as springs and fountains than in the wells (Table 1). Massa et al. (25) reported Aeromonas spp. in 25% of well water samples from Lecce, Italy. These data are similar to the results obtained in this study (30.3%), but lower than the 48.7% reported by Ghenghesh et al. (12) in Tripoli, Lybia.

 

 

The membrane filtration technique showed better results than the P/A method for recovery of Aeromonas spp. from the different water sources (Table 1). Considering that in both techniques the same sample volume (100 mL) and selective medium (ADA) are used, the lower percentage of positive samples obtained with P/A test could be attributed to the competitive flora in the P/A presumptive broth which is a medium favorable to the growth of different microbial indicators (9,10).

The counts of total and thermotolerant coliforms, heterotrophic bacteria and Aeromonas spp in the water samples are shown in Table 2. All the water sources presented maximum Aeromonas spp. counts higher than 200 CFU/100 mL. The fountain water samples showed the highest geometric mean and median for the bacterial indicators and Aeromonas spp., followed by the spring and well water samples. Aeromonas spp. was absent in the mineral water samples. However, the presence of more than 1.0x10³ CFU/mL of heterotrophic bacteria in these samples may have interfered in the detection of these microorganisms (2).

The different ranges of bacteriological indicators counts were compared with the positivity for Aeromonas spp. in order to determine a possible association between these bacteria and the routinely used pollution indicators (Table 3). Although the highest percentage of Aeromonas spp. positive samples was in samples with counts of total coliforms superior to 100 CFU/100 mL, Aeromonas spp. was also detected in well water samples where these indicators were absent. It was not observed an association between Aeromonas spp. and termotholerant coliforms, the opportunistic pathogen was commonly isolated in the absence of faecal contamination. Hirotani et al. (17) studied the correlation of A.hydrophila and fecal indicators and also observed that the presence of this microorganism was not associated with human fecal contamination. Araujo et al. (4) investigated a possible correlation between the presence of mesophilic aeromonads and the number of fecal coliforms and found a significant relationship only in polluted waters.

 

 

In the Netherlands, Aeromonas spp. are frequently present in drinking water in numbers varying between <1 and 1,0x104 CFU/100 mL and this organism can be isolated from 1-6% of faecal samples of patients with diarrhoea (16). In Italy, Aeromonas spp. were detected in water samples in counts ranging from 26 to 1609 CFU/250 mL (25).

The results of species identification are shown in Table 4. A.hydrophila was the most common species (48.3%), followed by A. allosacharophila (17.2%) and A. jandaei (10.3%). A. trota, A. sobria and HG 2 were also isolated. It was not possible to determine the species of ten out of 58 isolates submitted to the identification tests. These results are similar to the data reported in the literature, where A. hydrophila is the predominant species in freshwater and municipal drinking water supplies (12,22,31). The Aeromonas species that can cause human infections are A. hydrophila, A. caviae,A. veronii,A. jandaei,A. schubertii, A. trota and A. media (22,24). In the Northwest from Brazil, Aeromonas spp. were isolated from patients with diarrhea in a region where cholera cases were also occurring (Brazilian Health Department, personal communication).

 

 

Fifty-four strains of Aeromonas spp. were positive for haemolysin production. A thermolabile enterotoxin was detected in 13 out of 58 strains: A. hydrophila (n=6), A. jandaei (n= 3), A. allosacharophila (n=2) and Aeromonas sp (n=2).

There is little information available about toxin production by Aeromonas isolated from the environment (33,34). Some authors consider the b-hemolysis production as a virulence factor for Aeromonas species (7,33). In the present study, 22% of Aeromonas strains produced LT enterotoxin and 93% were haemolytic and can be therefore considered potentially enteropathogenic.

The data show that the raw drinking water sources studied represent a risk for human health and that commonly used bacteriological indicators cannot predict the presence of these potentially enteropathogenic microorganisms in such waters. Wells and springs are used as drinking water supply in poor areas and rural regions, where undernourished people more susceptible to infections by these microorganisms predominate, therefore disinfection practices and alternative sources of drinking water must be considered.

 

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* Corresponding Author. Mailing address: CETESB - Av. Prof. Frederico Hermann Jr. 345 cep 05459-900 São Paulo, SP - Brasil. Tel.: (11) 3133-3541 ou (11) 3133-3982. E-mail: mariaz@cetesbnet.sp.gov.br

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